HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources

Despite the high prevalence of both cervico-vaginal human papillomavirus (HPV) infection and bacterial vaginosis (BV) worldwide, their causal relationship remains unclear. While BV has been presumed to be a risk factor for HPV acquisition and related carcinogenesis for a long time, here, supported by both a large retrospective follow-up study (n = 6,085) and extensive in vivo data using the K14-HPV16 transgenic mouse model, we report a novel blueprint in which the opposite association also exists. Mechanistically, by interacting with several core members (NEMO, CK1 and β-TrCP) of both NF-κB and Wnt/β-catenin signaling pathways, we show that HPV E7 oncoprotein greatly inhibits host defense peptide expression. Physiologically secreted by the squamous mucosa lining the lower female genital tract, we demonstrate that some of these latter are fundamental factors governing host-microbial interactions. More specifically, several innate molecules down-regulated in case of HPV infection are hydrolyzed, internalized and used by the predominant Lactobacillus species as amino acid source sustaining their growth/survival. Collectively, this study reveals a new viral immune evasion strategy which, by its persistent/negative impact on lactic acid bacteria, ultimately causes the dysbiosis of vaginal microbiota.

peptides. a Representative pictures of morphologically normal ectocervix / TZ adjacent to CIN 2/3 stained for S100A7, elafin, HβD2, HD-5 and HD-6. b Semi-quantitative evaluation of innate peptide expression (intensity and extent of the immunostaining) in normal squamous epithelia from HPV-negative specimens (n=20, individual data from Figure 2F) and morphologically normal tissues adjacent to CIN 2/3 (n=20). The significantly reduced anti-S100A7, anti-elafin, anti-HβD2, anti-HβD4 and anti-HD-5 immunoreactivities should be noticed. Box limits: 25th to 75th percentiles; line: median; whiskers: minimum to maximum. The scale bar represents 100 μm. P values were determined using two-sided Mann-Whitney test. ns: not significant (p<0.1). Source data are provided as a Source Data file.

Supplementary Figure 4. Secretion of defensin-like peptides (SLPI, elafin, S100A7) and
HβD1 analyzed by ELISA. Note the drastic reduced secretion of these four so called "constitutive" peptides in case of HPV16 E6E7 transduction. Results represent the means ± SEM of three independent experiments. P values were determined using two-sided unpaired t-tests. Source data are provided as a Source Data file.

Supplementary Figure 5. Validation of HPV16 E7-NEMO, E7-CK1 and E7-β-TrCP interactions by co-IP in the inverse direction.
In contrast to the results presented in the main Figures 3 and 4, anti-NEMO, anti-CK1α1 and anti-β-TrCP antibodies were used for the immunoprecipitation step. FLAG-E7 was then detected. Note the clear enrichments supporting the bindings highlighted by GPCA. Each co-IP analysis was independently performed twice and one representative experiment is shown. Source data are provided as a Source Data file.

Supplementary Figure 6. Binding of NEMO with E7 oncoprotein from six different HPV genotypes (high-risk alpha: HPV16, 18, 33 and 39; beta: HPV8, 38 and 49) analyzed by GPCA (a) and confirmed by co-IP (b).
For the GPCA experiments, PTPN14 and PLEKHA9 were used as positive and negative control, respectively. Results represent the means ± SEM of two (PTPN14 and PLEKHA9) or four (NEMO) independent experiments. Each co-IP analysis was independently performed twice and one representative experiment is shown. P values were determined using twosided unpaired t-tests. Source data are provided as a Source Data file.

Supplementary Figure 7. Interaction between gamma isoforms (1-3) of CK1 and HPV16 E7
oncoprotein. a The bindings were validated by both Co-IP and GPCA using the truncated/mutated forms of E7. Of note, the GPCA signal was drastically reduced with the CR1+CR2 construct, supporting that CK11, CK12 and CK13 interact with the C-terminal region of E7. Results represent the means ± SEM of three independent experiments. b PTPN14 and Rb1 were systematically used as positive control for binding to the C-terminal region and the LxCxE motif (included in the N-terminal half) of E7, respectively. Results represent the means ± SEM of three independent experiments. Each co-IP analysis was independently performed twice and one representative experiment is shown. P values were determined using one-way ANOVA followed by Bonferroni post-hoc test. Source data are provided as a Source Data file. Figure 8. Co-IP of GSK3β with E7 oncoprotein from HPV16 and 18. Note the absence of clear enrichment suggesting that GSK3β represents a false positive of the GPCA screening method. Each co-IP analysis was independently performed twice and one representative experiment is shown. Source data are provided as a Source Data file. Figure 9. Binding of alpha and gamma isoforms of CK1 and β-TrCP with E7 oncoprotein from six different HPV genotypes (high-risk alpha: HPV16, 18, 33 and 39; beta: HPV8, 38 and 49) analyzed by GPCA (a) and confirmed by co-IP (b). For the GPCA experiments, PTPN14 and PLEKHA9 were used as positive and negative control, respectively. Results represent the means ± SEM of two (PTPN14 and PLEKHA9) or four (CK1 isoforms and β-TrCP) independent experiments. Each co-IP analysis was independently performed twice and one representative experiment is shown. P values were determined using two-sided unpaired ttests. ns: not significant (p<0.1). Source data are provided as a Source Data file. Figure 10. MALDI-TOF mass spectra of oxidized innate peptides incubated with or without DTT (2mM) and alkylated with iodoacetamide (IAA). In addition to the chemical reduction of the disulfide bridges, both the synthesis and the purity of each tested peptide was examined.